PROJECT SUMMARY Based on the best available evidence derived from human epidemiologic studies and animal research conducted in rodents and non-human primates, the U.S. Food and Drug Administration has issued a warning that repeated and/or lengthy exposures to anesthetic and sedative drugs between the third trimester and the third year of life may adversely affect brain development. The mechanism by which these short-acting drugs could have long-term consequences remains unclear. Nearly all previous research on this topic has been focused on neurons, however proper functioning of brain circuitry is also heavily dependent on other cell types. During fetal and postnatal development across mammalian species, oligodendrocytes (OLs) differentiate from oligodendrocyte precursor cells (OPCs) and generate myelin, which insulates axons to ensure high-fidelity transmission of electrical signals. Therefore, developing OLs represents a highly plausible and largely unexplored novel target for anesthetic neurotoxicity. We propose to test the central hypothesis that early developmental exposure to general anesthesia (GA) interferes with brain development by disrupting myelin formation. First, we will assess the overall impact of this hypothesis by determining which commonly used GAs interfere with myelination, identifying GA-induced neurologic deficits that result from impaired myelination, and testing the effects of GAs on human OL development. Our preliminary data suggests that GAs interfere with the function of OPCs, which are a pool of precursor cells that are critically important for developmental myelination. Thus, we will test the hypothesis that GAs act at the cellular level by inhibiting the survival, proliferation, differentiation, and/or homeostatic interactions of OPCs. Finally, we will test the hypothesis that GAs exert an epigenetic effect at the molecular level by inhibiting methylation of DNA sequences that regulate transcription of genes required for myelination. To address these questions, our study will employ innovative, cutting-edge experimental neuroscience tools, including human embryonic stem cells, conditional transgenic mice, two-photon in vivo real time imaging and cell ablation, and high throughput sequencing of the methylome. The experiments of this proposal are expected to define an entirely new mechanism of anesthetic toxicity. The adult nervous system recapitulates many elements of myelin development as a critical part of the defense against the devastating effects of demyelination disorders such as multiple sclerosis and amyotrophic lateral sclerosis and as a key feature of recovery from disabling injuries such as brain trauma and stroke. Thus, our findings will be significant for pregnant patients and young children, and also for the large population of adult patients with myelin-related pathology. The enhanced understanding of how anesthetics affect myelin formation gained here will allow researchers to design studies assessing the risks of anesthetic and/or sedative exposures in potentially vulnerable patients and to test preventative and treatment strategies to mitigate harm to those patients who are at risk.